Discovery of functional factorless internal ribosome entry site-like structures through virome mining

All viruses must co-opt the host translational machinery for viral protein synthesis. The dicistrovirus intergenic region internal ribosome entry site (IGR-IRES) utilizes the most streamlined translation mechanism by adopting a triple pseudoknot structure that directly recruits and binds within the intersubunit space of the ribosome and initiates translation from a non-AUG codon. The origin of this unprecedented mechanism is not known. Using a bioinformatics pipeline to examine the diversity and function of IRESs across RNA viromes, we searched for IRES-like RNA structures using RNA covariance models for multiple IRES sub-types, and tested functional IRES by using a dual-fluorescent lentiviral library reporter screen. We identified over >4,700 dicistro-like genomes with ~32% containing putative IRES structures, including novel viral genome arrangements with multiple IRESs and IRESs embedded within open-reading frames (ORFs). Predicted IRESs bound directly to purified ribosomes and supported internal ribosome entry activity in vitro and in vivo. Moreover, internal IRESs embedded within an ORF of monocistronic genomes were functional and operated simultaneously to produce the downstream ORF. We also identified IRES-like structures within non-dicistrovirus viral genomes, including in the families Tombusviridae and Narnaviridae that bound to ribosomes directly and a subset can direct internal ribosome entry. This study provides a framework to map the origin of factorless IRES mechanisms and study the diverse viral strategies utilizing RNA-based mechanisms.